Family of pain producing substances and methods to produce novel analgesic drugs

ABSTRACT

A method may include treating pain, shock, and/or inflammatory conditions in a subject. A method may include using a therapeutically effective amount of an antibody, a lipoxygenase inhibitor, a cytochrome P-450 enzyme inhibitor, and/or an antioxidant configurable to at least partially treat pain, shock, and/or inflammatory conditions in a subject. A method of treating pain, shock, and/or inflammatory conditions in a subject may include inactivating or preventing at least one linoleic acid metabolite to treat certain conditions (e.g., pain, shock, and/or inflammation).

PRIORITY CLAIM

This application is a divisional of U.S. patent application Ser. No.13/131,220 filed May 25, 2011 which claims priority to PCT/US2009/065739filed Nov. 24, 2009 also claims priority to U.S. Provisional ApplicationSer. No. 61/118,078 filed Nov. 26, 2008.

STATEMENT ON U.S. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under NIH Grant No. R01DA19585 awarded by the National Institutes of Health. Accordingly, theUnited States Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to the fields of treating apain, shock, and/or inflammatory condition in a subject. Morespecifically, the present invention is related to the use of apharmaceutical composition that comprises one or more compounds thatinhibit and/or minimize the production of oxidized linoleic acidmetabolites and/or block the activity of oxidized linoleic acidmetabolites.

2. Description of the Relevant Art

Many pain conditions are poorly managed by currently availableanalgesics. For example, burn injuries affect more than two millionpeople annually in the United States alone. Importantly, poor paincontrol in burn patients is known to increase the risk for long termadverse outcomes. This is a critical issue since surveys indicate thatabout one-half of burned patients have inadequate pain management.Patients suffering from a burn injury experience pain at the initialheat insult, during healing, and even in a chronic form, post burninjury.

Currently available analgesics for treating burn pain (eg., opiates,local anesthetics) demonstrate only limited efficacy and are associatedwith considerable adverse effects. In addition to burn pain, there aremany other pain states (e.g., inflammatory pain, neuropathic pain,cancer pain, herpes zoster pain, etc) for which currently availableanalgesics exhibit very limited activity, especially with repeateddosing.

Shock resulting from massive trauma, severe blood or fluid loss,systemic infections, insufficient cardiac output, or any other disorderor injury that leads to a hypoperfusional state is a serious, lifethreatening condition. Even with aggressive and prompt treatment shockis often fatal.

It is therefore desirable to develop a safer method of treating a pain,shock, and/or inflammatory condition in a subject.

SUMMARY

In some embodiments, one or more compounds that inhibit and/or minimizethe production of oxidized linoleic acid metabolites and/or block theactivity of oxidized linoleic acid metabolites may be used in thepreparation of a pharmaceutical composition for treating pain, shock,inflammatory conditions, or combinations thereof, in a mammal in needthereof.

A pharmaceutical composition for treating pain, shock, inflammatoryconditions, or combinations thereof in a subject may include one or morecompounds that inhibit and/or minimize the production of oxidizedlinoleic acid metabolites and/or block the activity of oxidized linoleicacid metabolites and one or more pharmaceutically acceptable carriers.

In some embodiments, the pharmaceutical composition comprises one ormore compounds that block the activity of oxidized linoleic acidmetabolites, wherein at least one of the compounds is an antibody thatbinds to at least one oxidized linoleic acid metabolite. Antibodies thatmay be used in the pharmaceutical composition include antibodies thatbind to a hydroxy linoleic acid metabolite, antibodies that bind to anepoxy linoleic acid metabolite, and antibodies that bind to an oxolinoleic acid metabolite. Examples of linoleic acid metabolites that theantibody binds to include, but are not limited to:(10E,12Z)-9-oxooctadeca-10,12-dienoic acid;(9Z,11E)-13-oxooctadeca-9,11-dienoic acid; 9-hydroxyoctadecadienoicacid; 13-hydroxyoctadecadienoic acid; 9(10)-dihydroxy-octadec-12-enoicacid; 12,13-dihydroxy-9Z-octadecenoic acid; (12Z)-9,10-epoxyoctadecenoicacid; 12,13-epoxyoctadec-9Z-enoic acid. The antibody may be a monoclonalantibody or a polyclonal antibody.

In an embodiment, the pharmaceutical composition may include one or morecompounds that inhibit and/or minimize the production of oxidizedlinoleic acid metabolites. In one embodiment, wherein at least one ofthe compounds that inhibit and/or minimize the production of oxidizedlinoleic acid metabolites is a cytochrome P-450 enzyme inhibitor.

In an embodiment, the pharmaceutical composition may include one or morecompounds that inhibit and/or minimize the production of oxidizedlinoleic acid metabolites. In one embodiment, at least one of thecompounds is an antioxidant sufficient to substantially inhibit and/orreduce the catalytic effect of relevant metabolic enzymes in theLinoleate pathway.

In an embodiment, a method of treating pain, shock, inflammatoryconditions, or combinations thereof in a subject includes administeringto a subject who would benefit from such treatment a therapeuticallyeffective amount of a pharmaceutical composition, wherein thepharmaceutical composition comprises one or more compounds that inhibitand/or minimize the production of oxidized linoleic acid metabolitesand/or block the activity of oxidized linoleic acid metabolites. Thepharmaceutical composition may be administered intravenously, orally,topically, or directly into the central nervous system.

BRIEF DESCRIPTION OF THE DRAWINGS

Advantages of the present invention may become apparent to those skilledin the art with the benefit of the following detailed description of thepreferred embodiments and upon reference to the accompanying drawingsdescribed herein below.

FIG. 1A depicts a graphical evaluation of the release of a TRPV1 agonistfrom superfusates of control and heated skin;

FIG. 1B depicts a graphical evaluation of the excitatory effect ofsuperfusates collected from heated skin when applied to TG neurons fromTRPV1 KO mice.

FIG. 1C depicts a bar graph summarizing the experimental results fromthe experiments of FIGS. 1A and 1B.

FIG. 1D depicts a bar graph of the results of pretreatment with theTRPV1 antagonist, iodo-resiniferatoxin (I-RTX, 200 nM), versus vehicleon [Ca²⁺]i levels after application of room temperature superfusatescollected from skin biopsies exposed to 48° C.

FIG. 1E depicts a time response curve of [Ca²⁺]i levels demonstratingthat heated skin evokes the release of compound(s) that activate CHOcells expressing TRPV1;

FIG. 1F depicts a bar graph of calcium accumulation evoked bycompound(s) released from heated skin in CHOs transfected with eitherTRPV1 or GFP constructs;

FIG. 1G depicts a comparison of calcium accumulation in cultured rat TGneurons evoked by various supernatants obtained from groups of skinbiopsies heated at various temperatures;

FIG. 1H depicts the effect of ipl injection of compound(s) isolated fromheated skin on spontaneous nocifensive behavior in WT vs TRPV1 KO mice;

FIG. 2 depicts a bar graph showing the effects of heat on the release ofoxidized linoleic acid metabolites in skin;

FIG. 3A depicts a graph demonstrating the increase in [Ca²⁺]i levels inTG neurons from WT mice evoked by 9-HODE;

FIG. 3B depicts a graph demonstrating the [Ca²⁺]i levels in TG neuronsfrom TRPV1 knockout (KO) mice after application of 9-HODE;

FIG. 3C depicts a bar graph comparing [Ca²⁺]i levels in TG neurons fromWT versus TRPV1 KO mice after application of 9-HODE;

FIG. 3D depicts a concentration-effect curve demonstrating thestimulatory effect of 9-HODE on iCGRP release from cultured rat TGneurons;

FIG. 3E depicts the effect of intraplantar (ipl) hindpaw injection of9-HODE on pain behavior in wild type (WT) vs TRPV1 KO mice;

FIG. 3F depicts a comparison of increased [Ca²⁺]i levels in TG neuronsfrom WT versus TRPV1 KO mice evoked by 9-oxoODE;

FIG. 3G depicts a comparison of increased [Ca²⁺]i levels in CHO cellsexpressing TRPV1 evoked by various linoleic acid metabolites;

FIG. 3H depicts a summary of increased [Ca²⁺]i levels in cultured TGneurons from WT mouse evoked by various linoleic acid metabolites;

FIG. 4A depicts a graph showing [Ca²⁺]i levels in TG neurons from WTmice evoked by 9,10-EpOME;

FIG. 4B depicts a graph showing [Ca²⁺]i levels in TG neurons from TRPV1knockout (KO) mice after application of 9,10-EpOME;

FIG. 4C depicts a bar graph showing [Ca²⁺]i levels in TG neurons from WTversus TRPV1 KO mice after application of 9,10-EpOME;

FIG. 4D depicts a comparison of [Ca²⁺]i levels in TG neurons afterapplication of either 9,10 ERpOME or 12, 13EpOME;

FIG. 4E depicts the effect of intraplantar (ipl) hindpaw injection of9,10-EpOME on pain behavior in wild type (WT) vs TRPV1 KO mice;

FIG. 4F depicts the effect of increased [Ca²⁺]i levels in CHO cellsexpressing either TRPV3 or TRPV4 after application of either 12,13 EpOMEor 9, 10EpOME;

FIG. 5A depicts a comparison of the effect of NDGA (10 uM), Indomethacin(COX inhibitor, 2 uM) and I-RTX (a TRPV1 antagonist, 200 nM) onheat-evoked iCGRP release from rat TG neurons;

FIG. 5B depicts a bar graph summarizing the effect of NDGA (30 uM) onheat (48° C.)-evoked inward current in rat TG neurons;

FIG. 5C depicts a bar graph summarizing the effect of miconazole (100uM) on heat (48° C.)-evoked inward current in rat TG neurons;

FIG. 5D depicts a bar graph demonstrating the effect of antibodiesagainst 9 and 13-HODE applied intracellularly at two differentconcentrations on heat (48° C.)-evoked inward current in rat TG neurons.

FIG. 6 depicts an schematic diagram of heat injury-evoked nociceptionand different intervention techniques.

FIG. 7 depicts a summary of activity of Linoleic acid metabolites onvarious TRP channels. The pathway is obtained from the website:http://www.genome.ad.jp/kegg/pathway/map/map00591.html

FIG. 8A depicts a graph of the release of endogenous TRPV1 agonists fromdepolarized spinal cords.

FIG. 8B depicts a comparison of change in [Ca²⁺]i evoked by superfusatefrom non-depolarized spinal cord and depolarized spinal cord in CHOcells expressing TRPV1;

FIG. 8C depicts a comparison of a change in [Ca²⁺]i evoked bysuperfusate from depolarized spinal cord in TG neurons from WT mice andTRPV1 knockout mice;

FIG. 8D depicts the change in the content of 9-HODE in the superfusatecaused by exposure of spinal cords to depolarizing solution;

FIG. 9A depicts a comparison of the effect of intrathecal administrationof Vehicle, a TRPV1 antagonist AMG 9810, and capsaicin with or withoutthe TRPV1 antagonist on tactile allodynia observed in the right hindpawof rats;

FIG. 9B depicts a comparison of the tactile allodynia evoked byintrathecal administration of 9-HODE in presence and absence of theTRPV1 antagonist AMG 9810 (50 ug);

FIG. 9C depicts a comparison of the peak tactile allodynia evoked bycapsaicin in presence and absence of AMG 9810;

FIG. 9D depicts a comparison of the peak tactile allodynia evoked by9-HODE in presence and absence of AMG 9810;

FIG. 10 depicts a comparison of antinociception for various treatmentsat a 15 minute time interval with demonstration of effects due tocompounds that block the synthesis of linoleic acid metabolites (eg.,NDGA) and compounds that block the actions of linoleic acid metabolites(Ab=antibodies);

FIG. 11 depicts the effect of various oxidized linoleic acid metaboliteson the activity of TRPA1 or TRPM8 ion channels;

FIG. 12 depicts the effect of lipids extracted from burned human skinsamples and applied to cultured trigeminal sensory neurons from control(wild type, “WT”) mice and from TRPV1 knock-out mice; and

FIG. 13 depicts the effect of lipids extracted from burned human skinsamples in CHO cells that expressed either TRPV1, TRPV2, TRPV3, TRPV4 orTRPA1.

FIG. 14 depicts the effect of linoleic acid to activate rat trigeminalsensory neurons from mice in the presence of cytochrome P450 inhibitors,nitric oxide synthase inhibitors, lipoxygenase inhibitors andantioxidants.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof are shown by way ofexample in the drawings and may herein be described in detail. Thedrawings may not be to scale. It should be understood, however, that thedrawings and detailed description thereto are not intended to limit theinvention to the particular form disclosed, but on the contrary, theintention is to cover all modifications, equivalents and alternativesfalling within the spirit and scope of the present invention as definedby the appended claims.

DETAILED DESCRIPTION

It is to be understood the present invention is not limited toparticular devices or biological systems, which may, of course, vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a”, “an”, and “the” include singular and pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “a linker” includes one or more linkers.

Definitions

The terms used throughout this specification generally have theirordinary meanings in the art, within the context of the invention, andin the specific context where each term is used. Certain terms arediscussed below, or elsewhere in the specification, to provideadditional guidance to the practitioner in describing the devices andmethods of the invention and how to make and use them. It will beappreciated that the same thing can be said in more than one way.Consequently, alternative language and synonyms may be used for any oneor more of the terms discussed herein, nor is any special significanceto be placed upon whether or not a term is elaborated or discussed ingreater detail herein. Synonyms for certain terms are provided. Arecital of one or more synonyms does not exclude the use of othersynonyms. The use of examples anywhere in this specification, includingexamples of any terms discussed herein, is illustrative only, and in noway limits the scope and meaning of the invention or of any exemplifiedterm.

As used herein the terms “administration,” “administering,” or the like,when used in the context of providing a pharmaceutical or nutraceuticalcomposition to a subject generally refers to providing to the subjectone or more pharmaceutical, “over-the-counter” (OTC) or nutraceuticalcompositions in combination with an appropriate delivery vehicle by anymeans such that the administered compound achieves one or more of theintended biological effects for which the compound was administered. Byway of non-limiting example, a composition may be administered byparenteral, subcutaneous, intravenous, intracoronary, rectal,intramuscular, intra-peritoneal, transdermal, or buccal routes ofdelivery. Alternatively, or concurrently, administration may be by theoral route. The dosage administered will be dependent upon the age,health, weight, and/or disease state of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and/or the natureof the effect desired. The dosage of pharmacologically active compoundthat is administered will be dependent upon multiple factors, such asthe age, health, weight, and/or disease state of the recipient,concurrent treatments, if any, the frequency of treatment, and/or thenature and magnitude of the biological effect that is desired.

As used herein, the term “agonist” generally refers to a type of ligandor drug that binds and alters the activity of a receptor.

As used herein, the term “antagonist” generally refers to a type ofreceptor ligand which binds a receptor but which does not alter theactivity of the receptor; however when used with an agonist, preventsthe binding of the agonist to the receptor hence the effect of theagonist.

As used herein, the term “allodynia” generally refers to pain fromstimuli which are not normally painful. The pain may occur other than inthe area stimulated. Allodynia may generally refer to other pain.

As used herein, the term “antinociception” generally refers to areduction in pain sensitivity.

As used herein, the term “monoclonal antibody” generally refers to anantibody obtained from a population of substantially homogeneousantibodies (the individual antibodies comprising the population areidentical except for possible naturally occurring mutations that may bepresent in minor amounts). As used herein, the term “polyclonalantibody” generally refers to a population of antibodies that aredirected against a common epitope but which are not identical instructure.

As used herein, terms such as “pharmaceutical composition,”“pharmaceutical formulation,” “pharmaceutical preparation,” or the like,generally refer to formulations that are adapted to deliver a prescribeddosage of one or more pharmacologically active compounds to a cell, agroup of cells, an organ or tissue, an animal or a human. Methods ofincorporating pharmacologically active compounds into pharmaceuticalpreparations are widely known in the art. The determination of anappropriate prescribed dosage of a pharmacologically active compound toinclude in a pharmaceutical composition in order to achieve a desiredbiological outcome is within the skill level of an ordinary practitionerof the art. A pharmaceutical composition may be provided assustained-release or timed-release formulations. Such formulations mayrelease a bolus of a compound from the formulation at a desired time, ormay ensure a relatively constant amount of the compound present in thedosage is released over a given period of time. Terms such as “sustainedrelease” or “timed release” and the like are widely used in thepharmaceutical arts and are readily understood by a practitioner ofordinary skill in the art. Pharmaceutical preparations may be preparedas solids, semi-solids, gels, hydrogels, liquids, solutions,suspensions, emulsions, aerosols, powders, or combinations thereof.Included in a pharmaceutical preparation may be one or more carriers,preservatives, flavorings, excipients, coatings, stabilizers, binders,solvents and/or auxiliaries that are, typically, pharmacologicallyinert. It will be readily appreciated by an ordinary practitioner of theart that, pharmaceutical compositions, formulations and preparations mayinclude pharmaceutically acceptable salts of compounds. It will furtherbe appreciated by an ordinary practitioner of the art that the term alsoencompasses those pharmaceutical compositions that contain an admixtureof two or more pharmacologically active compounds, such compounds beingadministered, for example, as a combination therapy.

As used herein the term “pharmaceutically acceptable salts” includessalts prepared from by reacting pharmaceutically acceptable non-toxicbases or acids, including inorganic or organic bases, with inorganic ororganic acids. Pharmaceutically acceptable salts may include saltsderived from inorganic bases include aluminum, ammonium, calcium,copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous,potassium, sodium, zinc, etc. Examples include the ammonium, calcium,magnesium, potassium, and sodium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, and basic ionexchange resins, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-dibenzylethylenediamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine, etc.

The terms “reducing,” “inhibiting” and “ameliorating,” as used herein,when used in the context of modulating a pathological or disease state,generally refers to the prevention and/or reduction of at least aportion of the negative consequences of the disease state. When used inthe context of an adverse side effect associated with the administrationof a drug to a subject, the term(s) generally refer to a net reductionin the severity or seriousness of said adverse side effects.

As used herein the term “subject” generally refers to a mammal, and inparticular to a human.

As used herein, the term “treat” generally refers to an action taken bya caregiver that involves substantially inhibiting, slowing or reversingthe progression of a disease, disorder or condition, substantiallyameliorating clinical symptoms of a disease disorder or condition, orsubstantially preventing the appearance of clinical symptoms of adisease, disorder or condition.

Terms such as “in need of treatment,” “in need thereof,” “benefit fromsuch treatment,” and the like, when used in the context of a subjectbeing administered a pharmacologically active composition, generallyrefers to a judgment made by an appropriate healthcare provider that anindividual or animal requires or will benefit from a specified treatmentor medical intervention. Such judgments may be made based on a varietyof factors that are in the realm of expertise of healthcare providers,but include knowledge that the individual or animal is ill, will be ill,or is at risk of becoming ill, as the result of a condition that may beameliorated or treated with the specified medical intervention.

By “therapeutically effective amount” is meant an amount of a drug orpharmaceutical composition that will elicit at least one desiredbiological or physiological response of a cell, a tissue, a system,animal or human that is being sought by a researcher, veterinarian,physician or other caregiver.

Methods and Compositions

Mechanistically, burn injury is a unique type of tissue damage wheretransient exposure to heat results in long lasting changes in theexposed tissue (e.g., skin). The studies performed in humans and inanimals demonstrated that these changes in the damaged tissue are atleast in part responsible for the development and maintenance of ongoingpain or hyperalgesia. The heat injured tissue generates variousinflammatory mediators that sensitize ion channels such as transientreceptor potential vanilloid 1 or TRPV1 to evoke ongoing pain andhyperalgesia. Other important pain conditions include inflammatory,neuropathic, cancer and the like.

TRPV1, also known as the capsaicin receptor, plays a pivotal role inburn injury and other important pain conditions by evoked hyperalgesiaand allodynia such that the mice deficient in TRPV1 protein show littleto no hyperalgesia in these models. The key role played by TRPV1 in thedevelopment of thermal hyperalgesia and possibly mechanical hyperalgesiain various pain models is well established in animal and human studies.Signaling cascades initiated by a variety of inflammatory mediators maysensitize TRPV1 and contribute to inflammatory hyperalgesia. Given theimportance of TRPV1 in inflammatory pain, burn pain and cancer pain,including other various pain states, antagonists against TRPV1 may beused for treating pain and/or inflammatory conditions. However, recentstudies have demonstrated some serious on target side effects of TRPV1antagonists that may exclude their clinical use. These data necessitateadditional research in findings ways to block TRPV1 activation withoutusing the antagonists.

A variety of endogenous molecules have been shown to activate TRPV1 andthey include anandamide, N-arachidonoyl-dopamine, N-oleoyldopamine,polyamines etc. Such endogenous TRPV1 ligands may be generated duringinflammation and contribute to constitutive activation of TRPV1. Barringa few reports, the role of these endogenous TRPV1 ligands inphysiological or pathological pain is not known. Interestingly, TRPV1may be activated by stimuli such as protons and noxious heat. Themechanism by which heat activates TRPV1 is not completely understoodalthough several hypotheses have been proposed. Heat may generateendogenous TRPV1-stimulating ligands in the heat-exposed tissue and thusinitiates noxious pain sensation. In tissues exposed to heat for longerdurations, the endogenous TRPV1 ligands may be constitutivelysynthesized and activate TRPV1 to produce ongoing pain sensation in theabsence of heat. A similar pathway may exist for inflammatory or otherpain conditions as well.

In some embodiments, heat injury to organs such as skin results ingeneration of oxidized linoleic acid metabolites. These metabolitesrepresent a novel family of endogenous TRPV1 ligands. These ligandsactivate TRPV1 expressed by sensory nerve terminals in the damagedtissue. The opening of TRPV1 leads to generation of action potentialsand the pain sensation in the somatosensory cortex.

In some embodiments, metabolites of linoleic acid have been identifiedas TRPV1 agonists. Linoleic acid is also known by its IUPAC name cis,cis-9,12-octadecadienoic acid. Linoleic acid has a structure:

In some embodiments, pharmacological interventions that can block thegeneration of the endogenous TRPV1 ligand in response to heat may be oftherapeutic use. FIG. 7 depicts a summary of activity of Linoleic acidmetabolites on various TRP channels and ways of interrupting activity ofthe metabolites on these channels. In addition, the measurement ofLinoleic acid metabolites may constitute a novel method for diagnosingpain or shock conditions, thereby guiding treatment selection.

In some embodiments, oxidized linoleic acid metabolites are generatedupon heat stimulation of skin. Oxidized linoleic acid metabolitesinclude, but are not limited to, oxo linoleic acid metabolites, hydroxyllinoleic acid metabolites, and epoxy linoleic acid metabolites. Examplesof oxo linoleic acid metabolites include, but are not limited to(10E,12Z)-9-oxooctadeca-10,12-dienoic acid (9-oxoODE, 9-KODE) and(9Z,11E)-13-oxooctadeca-9,11-dienoic acid (13-oxoODE, 13-KODE). Examplesof hydroxyl linoleic acid metabolites include, but are not limited to:9-hydroxyoctadecadienoic acid (9-HODE); 13-hydroxyoctadecadienoic acid(13-HODE); 9(10)-dihydroxy-octadec-12-enoic acid (9,10-DiHOME); and12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME). Examples of epoxylinoleic acid metabolites include, but are not limited to:(12Z)-9,10-epoxyoctadecenoic acid (9(10)-EpOME) and12,13-epoxyoctadec-9Z-enoic acid (12(13)-EpOME). It is believed thatoxidized linoleic acid metabolites may function as endogenous TRPV1agonists.

In some embodiments, the blockade of synthesis or immunoneutralizationof oxidized linoleic acid metabolites results in decreased activation ofpain sensing neurons by heat in vitro and results in thermalantinociception in vivo. Immunoneutralization of oxidized linoleic acidmetabolites may be accomplished by the use of one or more antibodiesthat bind to at least one oxidized linoleic acid metabolite. Antibodiesfor oxidized linoleic acids may be formed using the procedure ofSpindler et al. (Spindler et al. “Significance and immunoassay of 9- and13-hydroxyoctadecadienoic acids.” Biochem Biophys Res Commun. 1996;218:187-191), which is incorporated herein by reference.

TRPV1 is not the only heat-sensitive channel in the body. In someembodiments, other members of the family, TRPV3 and TRPV4 are activatedby warm temperatures. Therefore, the linoleic acid metabolites werescreened against these channels In some embodiments, 12,13-EpOME and9,10-DiHOME are TRPV3 and TRPV4 agonists respectively.

FIG. 6 depicts an embodiment of a model of heat injury-evokednociception and different intervention embodiments.

In some embodiments, intrathecal application of nordihydroguaiareticacid (NDGA) or neutralizing antibodies against 9-HODE and 13-HODE may bean effective way to block inflammatory allodynia. Thus compounds such asNDGA and neutralizing antibodies may have two unique sites of action inthe treatment of thermal and mechanical allodynia. NDGA is alipoxygenase (LOX) inhibitor and an antioxidant. LOX inhibitors may beadministered sufficiently to substantially attenuate the catalyticeffect of enzymes such as EC 1.13.11.34 (aka: arachidonate5-lipoxygenase) in order to treat pain, shock, and/or inflammatoryconditions. In some embodiments, LOX inhibitors other than NDGA may beadministered.

In some embodiments, a method of treating a pain, shock and/orinflammatory conditions may include administering a cytochrome P-450(CYP) enzyme inhibitor sufficient to substantially inhibit and/or reducethe catalytic effect of multiple P450 isozymes capable of synthesizingoxidized linoleic acid metabolites (OLAMs). In some embodiments, the CYPinhibitor may be administered intravenously, orally, topically (forburns or wounds), directly into the central nervous system (e.g.,epidural), or any other method described herein or that will be known tothose skilled in the art.

In some embodiments, a method of treating a pain, shock and/orinflammatory conditions may include administering a cytochrome P-450(CYP) isoenzyme inhibitor sufficient to substantially inhibit or reducethe catalytic effect of enzyme EC 1.14.14.1 (aka: CYP2C9 and CYP2C 19).

Examples of CYP inhibitors include, but are not limited to;ketoconazole, micronazole, fluconazole, benzbromarone, sulfaphenazole,valproic acid, amiodarone, cimetidine, fenofibrate, fluvastatin,lovastatin, fluvoxamine, sertraline, isoniazid, probenecid,sulfamethoxazole, teniposide, voriconazole, and zafirlukast. In someembodiments, the CYP inhibitor may be administered intravenously,orally, topically (for burns or wounds), directly into the centralnervous system (e.g., epidural), or any other method described herein orthat will be known to those skilled in the art.

In one embodiment, cytochrome P450 inhibitors that block the formationof linoleic acid metabolites may be used as analgesic drugs. In oneembodiment, ketoconazole is administered topically or systemically torelieve pain or inflammation, shock or hypotension mediated by theformation of linoleic acid metabolites.

In some embodiments, a method of treating a pain, shock, and/orinflammatory condition may include administering an antioxidantsufficient to substantially inhibit and/or reduce the catalytic effectof relevant metabolic enzymes in the Linoleate pathway. In someembodiments, antioxidant inhibitors of relevant metabolic enzymes in theLinoleate pathway may include Nordihydroguaiaretic acid (NDGA), VitaminE and/or Vitamin E derivatives (e.g., water soluble Vitamin Ederivative). NDGA may function at least in part as a therapeutic agentdue to its strong antioxidant characteristics.

Recent research has indicated that activation of TRPV1 by 9-HODE mayhave other roles in the body depending upon the expression of TRPV1.TRPV1 in the spinal cord may play an important role in maintenance ofthermal and mechanical allodynia in inflammatory or other painconditions. Depolarization of the spinal cord may lead to the release of9-HODE and activation of TRPV1. 9-HODE in the spinal cord may lead todevelopment of mechanical allodynia. Similar to heated skin, depolarizedspinal cord (with high potassium) may release compound(s) that haveTRPV1 agonist activity. Depolarized spinal cord superfusate may containsignificantly higher amounts of 9-HODE. Moreover, activation of TRPV1 inthe spinal cord by capsaicin (positive control) or by 9-HODE results intactile allodynia that is completely reversible by a TRPV1 antagonist.Thus, in some embodiments, the role of 9-HODE and similar linoleic acidoxidation products extends beyond heat-nociception.

In some embodiments, a method may include treating shock and/orinflammation. The therapy used to treat any one case of shock dependsupon the cause of the patient's hypoperfusional disorder, however, adisruption in cellular membrane integrity, leading to the release andoxidation of linoleic acid metabolites from stressed cells, is a processcommon to many if not most cases of shock. These oxidized linoleic acidmetabolites have paracrine and/or endocrine effects that act to worsenthe symptoms of shock. A method as described herein may effectivelydelay the multi-organ failure associated with Refractory (Irreversible)shock. This therapeutic method may be used in many, if not most cases ofshock and save many lives.

In some embodiments, given the role of these metabolites in variousother diseases (e.g., arthritis, pulmonary edema and shock), similarmethods and antibodies may be used in treating these conditions.

Any suitable route of administration may be employed for providing asubject with an effective dosage of the compositions (e.g., antibodies,compounds) described herein. For example, oral, rectal, topical,parenteral, ocular, pulmonary, nasal, and the like may be employed.Dosage forms include tablets, troches, dispersions, suspensions,solutions, capsules, creams, ointments, aerosols, and the like.

The compositions may include those compositions suitable for oral,rectal, topical, parenteral (including subcutaneous, intramuscular, andintravenous), ocular (ophthalmic), pulmonary (aerosol inhalation), ornasal administration, although the most suitable route in any given casewill depend on the nature and severity of the conditions being treatedand on the nature of the active ingredient. They may be convenientlypresented in unit dosage form and prepared by any of the methodswell-known in the art of pharmacy.

In practical use, compositions may be combined as the active ingredientin intimate admixture with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques. The carrier may takea wide variety of forms depending on the form of preparation desired foradministration, e.g., oral or parenteral (including intravenous). Inpreparing the compositions for oral dosage form, any of the usualpharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols, flavoring agents, preservatives, coloringagents and the like in the case of oral liquid preparations, such as,for example, suspensions, elixirs and solutions; or carriers such asstarches, sugars, microcrystalline cellulose, diluents, granulatingagents, lubricants, binders, disintegrating agents and the like in thecase of oral solid preparations such as, for example, powders, capsulesand tablets, with the solid oral preparations being preferred over theliquid preparations. Because of their ease of administration, tabletsand capsules represent the most advantageous oral dosage unit form inwhich case solid pharmaceutical carriers are obviously employed. Ifdesired, tablets may be coated by standard aqueous or nonaqueoustechniques.

The pharmaceutical preparations may be manufactured in a manner which isitself known to one skilled in the art, for example, by means ofconventional mixing, granulating, dragee-making, softgel encapsulation,dissolving, extracting, or lyophilizing processes. Thus, pharmaceuticalpreparations for oral use may be obtained by combining the compositionswith solid and semi-solid excipients and suitable preservatives, and/orco-antioxidants. Optionally, the resulting mixture may be ground andprocessed. The resulting mixture of granules may be used, after addingsuitable auxiliaries, if desired or necessary, to obtain tablets,softgels, lozenges, capsules, or dragee cores.

Suitable excipients may be fillers such as saccharides (e.g., lactose,sucrose, or mannose), sugar alcohols (e.g., mannitol or sorbitol),cellulose preparations and/or calcium phosphates (e.g., tricalciumphosphate or calcium hydrogen phosphate). In addition binders may beused such as starch paste (e.g., maize or corn starch, wheat starch,rice starch, potato starch, gelatin, tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidone). Disintegrating agents may be added (e.g., theabove-mentioned starches) as well as carboxymethyl-starch, cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof (e.g.,sodium alginate). Auxiliaries are, above all, flow-regulating agents andlubricants (e.g., silica, talc, stearic acid or salts thereof, such asmagnesium stearate or calcium stearate, and/or polyethylene glycol, orPEG). Dragee cores are provided with suitable coatings, which, ifdesired, are resistant to gastric juices. Soft gelatin capsules(“softgels”) are provided with suitable coatings, which, typically,contain gelatin and/or suitable edible dye(s). Animal component-free andkosher gelatin capsules may be particularly suitable for the embodimentsdescribed herein for wide availability of usage and consumption. Forthis purpose, concentrated saccharide solutions may be used, which mayoptionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethyleneglycol (PEG) and/or titanium dioxide, lacquer solutions and suitableorganic solvents or solvent mixtures, including dimethylsulfoxide(DMSO), tetrahydrofuran (THF), acetone, ethanol, or other suitablesolvents and co-solvents. In order to produce coatings resistant togastric juices, solutions of suitable cellulose preparations such asacetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate,may be used. Dye stuffs or pigments may be added to the tablets ordragee coatings or soft gelatin capsules, for example, foridentification or in order to characterize combinations of activecompound doses, or to disguise the capsule contents for usage inclinical or other studies.

In some embodiments, compositions (e.g., antibodies) will typically beformulated in such vehicles at a concentration of about 0.1 mg/ml to 100mg/ml.

For the prevention or treatment of disease, the appropriate dosage ofthe composition will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether thecompositions are administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to thecomposition, and the discretion of the attending physician. Thecomposition is suitably administered to the patient at one time or overa series of treatments.

Depending on the type and severity of the disease, about 0.015 to 15mg/kg of composition (e.g., antibodies) is an initial candidate dosagefor administration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. For repeatedadministrations over several days or longer, depending on the condition,the treatment is repeated until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful.

According to another embodiment of the invention, the effectiveness ofthe composition in preventing or treating disease may be improved byadministering the composition serially or in combination with anotheragent that is effective for those purposes, such as another antibodydirected against a different epitope or neutralizing a different proteinthan the first composition, or one or more conventional therapeuticagents such as, for example, alkylating agents, folic acid antagonists,anti-metabolites of nucleic acid metabolism, antibiotics, pyrimidineanalogs, 5-fluorouracil, purine nucleosides, amines, amino acids,triazol nucleosides, corticosteroids, calcium, retinoids, lipoxygenaseand cyclooxygenase inhibitors, fumaric acid and its salts, analgesics,psychopharmaceuticals, local anesthetics, spasmolytics, andbeta-blockers. Such other agents may be present in the composition beingadministered or may be administered separately. The composition may besuitably administered serially or in combination with radiologicaltreatments, whether involving irradiation or administration ofradioactive substances.

EXAMPLES

FIGS. 1A-H depict that endogenous TRPV1 ligands are generated upon heatinsult of mouse skin. The hypothesis of heat-evoked generation ofendogenous TRPV1 ligand(s) was evaluated by harvesting mouse skin andthen collecting superfusate samples after 20 min exposure to basal (37°C.=“control” skin) or noxious (48° C.=“heated” skin) temperatures.Aliquots of the two superfusates were applied, at room temperature, tocultured trigeminal ganglia (TG) neurons from wild type (WT) or TRPV1knock out (TRPV1 KO) mice for measurement of evoked increases inintracellular calcium levels [Ca²⁺]i. In contrast to the supernatantscollected from skin exposed to basal temperatures, the application ofsupernatants collected from heated skin demonstrated a rapid increase in[Ca²⁺]i, but only in those WT neurons that were also capsaicin-sensitive(FIG. 1A). Conversely, there were no changes in [Ca²⁺]i afterapplication of supernatants from heated skin to either TRPV1 KO cultures(FIG. 1B; mustard oil served as a positive control) or to WT neuronsinsensitive to capsaicin (data not shown). These data are summarized inFIG. 1C, which indicates that heated skin selectively releasescompound(s) that activate TRPV1. Interestingly, superfusate from skinsexposed to non-noxious temperature did not contain any substance(s) thatactivated TG neurons (FIG. 1G). An independent series of controlexperiments demonstrated that supernatants obtained from heated plasticculture wells (i.e., no skin biopsies) did not alter [Ca²⁺]i in TGneurons, demonstrating that these liberated compounds were of biologicalorigin (data not shown). Initial characterization studies demonstratedthat these compound(s) were unstable at room temperature (>4 h), butcould be isolated from C₁₈ reverse phase columns using a step gradientof 0-90% acetonitrile; this dried down fraction (N₂ gas) was stable at−80° C. for prolonged periods and formed the pool of endogenouscompound(s) used in subsequent studies.

To further characterize the TRPV1 specificity of these endogenousheat-generated compound(s), the effect of pretreatment with the TRPV1antagonist iodo-resiniferatoxin (I-RTX) on the activation of rat TGneurons was evaluated. I-RTX completely blocked the activation ofneurons by compound(s) released from heated skin (FIG. 1D). Moreover,application of these compound(s) triggered prompt increases in [Ca]i inCHO cells expressing TRPV1, but not in negative control CHO cells thatexpressed a GFP construct (FIGS. 1E-F). Next, initial superfusionexperiments in isolated mouse skin were expanded by collectingsupernatant after exposure to various temperatures and evaluating therelease of endogenous TRPV1 compound(s). Such activity was observed insupernatants collected only from skin biopsies exposed to noxioustemperatures (43° C. and 48° C.) and not in skin samples exposed tonon-noxious temperatures (22-37° C.) (FIG. 1G). Finally, the C₁₈isolated compounds produced significantly greater nocifensive behaviorwhen injected ipl into the hindpaws of WT mice compared with TRPV1 KOmice (FIG. 1H).

FIG. 2 depicts that heat evokes the release of oxidized linoleic acidmetabolites from isolated and superfused skin biopsies. The enzymeimmunoassay (EIA) analyses of compounds released into mouse skinsuperfusates demonstrated an increased presence of the linoleic acidoxidation products 9-HODE in the heated skin samples, compared with thenon-heated samples (FIG. 2). The EIA finding is important fordemonstrating the detection of μM quantities (˜3 μg in 1 ml) of released9-HODE when assayed immediately after collection.

FIGS. 3A-H depict that lipoxy linoleic acid metabolites are TRPV1agonists. The pharmacological activity of synthetic 9-HODE and relatedcompounds on sensory neurons was characterized. Application of 9-HODE(100 μM) evoked a robust response in TG neurons cultured from WT mice(FIG. 3A), but not in neurons from TRPV1 KO mice (FIG. 3B, 50 mMpotassium was positive control to verify neuronal viability). Detailedanalysis of multiple experiments revealed that the neuronal populationthat responded to 9-HODE in WT mice coincided completely (75/75) withthe capsaicin-sensitive subpopulation of neurons. The application of9-HODE to neurons cultured from TRPV1 KO mice revealed a slight responsein 4% of tested neurons (3/66); however, both the magnitude of [Ca²⁺]iaccumulation and its time course were greatly reduced compared to WTneurons (FIG. 3C). The application of 9-HODE to cultured rat TG neuronsproduced a concentration-dependent increase in CGRP release, witheffects evident at 10 nM and an EC₅₀˜300 nM (FIG. 3D). Using observersblinded to treatment allocation, hindpaw intraplantar (ipl) injectionsof 9-HODE produced significantly greater thermal allodynia in WT micebut not in TRPV1 KO mice (FIG. 3E). Application of 9-oxoODE, ametabolite of 9-HODE, also triggered increased [Ca²⁺]i levels, but onlyin TG neurons from WT mice and not in neurons from TRPV1 KO mice (FIG.3F). Another major metabolic pathway of linoleic acid leads to formationof 13-HODE and its metabolite, 13-oxoDE. The relative efficacy of thesefour compounds for evoked [Ca²⁺]i levels was evident when tested incultured neurons from WT mice (FIG. 3G) and in TRPV1-expressing CHOcells (FIG. 3H).

FIGS. 4A-F depict that epoxy linoleic acid metabolites are also TRPV1agonists. The epoxy metabolite, 9,10 EpOME (also known as leukotoxin),is highly elevated in burn patients and is implicated in shock in thesepatients. Similar to the previous studies on the HODEs, the applicationof 9, 10 EpOME (100 μM) evoked calcium accumulation only incapsaicin-sensitive neurons (58/58) from WT mice (FIG. 4A), but not fromTRPV1 KO mice (0/58) (FIG. 4B). A comparison of respective responses isdemonstrated in FIG. 4C. A parallel epoxy metabolite, 12, 13 EpOME, wasalso found to be a TRPV1 agonist and relative efficacy in CHO cellsexpressing TRPV1 is shown in FIG. 4D. 9, 10 EpOME (50 μg/ipl) evokedthermal allodynia in WT mice that was absent in TRPV1 KO mice (FIG. 4E).These data demonstrate that TRPV1 activating activity of linoleic acidproducts extends to epoxy products as well. Upon screening of thesemetabolites against other heat-sensitive TRP channels, it was discoveredthat 12,13-EpOME is a TRPV3 agonist and 9,10-DiHOME is a TRPV4 agonist.

FIGS. 5A-D depict that interventions reducing production or action oflinoleic acid oxidation products results in decreased thermal pain.Whether heat-evoked activation of rat TG neurons is inhibited byinterventions directed against linoleic acid metabolites was evaluated.Since oxidized linoleic acid metabolites can be formed eitherenzymatically (via lipoxygenase) or spontaneously (via free radicals),nordihydroguaiaretic acid (NDGA) was selected to block their synthesissince this compound inhibits both pathways. In a CGRP release assay,heat-evoked (48° C.) iCGRP release was partially reversed bypretreatment with NDGA and I-RTX but not by a COX-inhibitor indomethacin(FIG. 5A). Using patch clamp electrophysiology, the local application ofa heat step (48° C.) produced a strong inward current (I_(heat)) in TGneurons. Pretreatment with NDGA produced ˜50% reduction (p<0.05) inI_(heat). (FIG. 5B). In a similar patch clamp electrophysiology set up,pretreatment with miconazole (100 uM) produced more than 75% reduction(p<0.001) in I_(heat) (FIG. 5C). Whether intracellularimmunoneutralization against 9-HODE and 13-HODE alters theresponsiveness of sensory neurons to noxious heat was evaluated next. Asolution containing both anti-9-HODE and anti-13-HODE antibodies(concentrations of 0.03-0.06 ug and 0.006-0.012 ug/patch pipetterespectively) was delivered intracellularly via dialysis for 5 min priorto the application of a heat step (48° C.). This treatment combinationsignificantly decreased I_(heat) by up to 54% (p<0.05; FIG. 5D).Dialysis of each antibody alone did not significantly reduce I_(heat),implicating a redundancy in the signaling properties of these compounds.

FIGS. 8A-D depict that depolarization of the spinal cord leads torelease of endogenous TRPV1 ligand(s) that coincides with increased9-HODE in the superfusate. TRPV1 in the spinal cord is criticallyinvolved in the maintenance of inflammatory hyperalgesia/allodynia. Thissuggests that TRPV1 in the spinal cord is tonically activated ininflammatory conditions. One possibility is that ongoing discharge inthe primary afferent neurons leads to the release of an endogenous TRPV1ligand in the spinal cord and this ligand in tern tonically activatesTRPV1. To test this hypothesis, freshly isolated rat spinal cords (9)was depolarized using 50 mM potassium and the resulting superfusateswere purified using C₁₈ columns similar to the previous skin experimentsdescribed herein above. Purified depolarized spinal cord superfusateactivated CHO cells transfected with TRPV1, but purified superfusateobtained from the same spinal cords under basal (non-depolarized)conditions was unable to do so (FIGS. 8A-B). The specificity of thedepolarized superfusate was further confirmed by its inability toactivate any neurons from TRPV1 KO mice in contrast to a robust responsein neurons from WT mice (FIG. 8C). EIA analysis demonstrated thatdepolarized spinal cord superfusate had a significantly higher 9-HODEcontent than the non-depolarized superfusate (FIG. 8D). Thus, theoxidized linoleic acid metabolites are released from both peripheraltissues and central neurons.

FIGS. 9A-D depict that spinal injection (intrathecal) of capsaicin or9-HODE into the spinal cord space results in tactile allodynia. Thiseffect is mediated by activation of TRPV1 since it is blocked by a TRPV1antagonist (AMG 9810). Since it has been established that a depolarizedspinal cord contains significantly greater amounts of 9-HODE, theability of 9-HODE to evoke tactile allodynia upon intrathecalapplication was next evaluated. The results were compared with theresults obtained with the positive control capsaicin. Indeed, in rats,intrathecal application of capsaicin (5 ug) evoked tactile allodyniathat lasted 1 hour post injection and was completely reversible by aTRPV1 antagonist AMG 9810 (50 ug, FIG. 9A). Neither the vehicle nor AMG9810 alone had any effect on mechanical withdrawal thresholds in rats.The intrathecal injection of synthetic 9-HODE at the same dose (5 ug)evoked tactile allodynia that was of similar magnitude and duration tothat of capsaicin. Moreover, the effect of 9-HODE was also completelyreversible by AMG 9810, suggesting exclusive involvement of TRPV1 inmediating the effect (FIG. 9B). Comparison of the peak effect evoked byboth capsaicin and 9-HODE is shown in FIGS. 9C-D. The effects wereremarkably similar.

FIG. 10 depicts the behavioral significance of blockade of linoleic acidoxidation on thermal nociception in rats. The intraplantar hindpaw (ipl)injection of NDGA, miconazole, and a combination of anti 9-HODE andanti-13-HODE was compared to morphine sulfate, a gold standardanalgesic. The examined doses only produced a locally mediated effect,since the responses were only observed in the ipsilateral (injected)hindpaw and not in the contralateral paw. Under these conditions,miconazole, NDGA and the antibodies all produced a thermalantinociception far beyond that of the vehicle or morphine injectedrats. The studies were performed in a separate groups of animals foreach condition and by observers blinded to treatment allocation (allcompounds at 100 ug/paw and antibodies were each 25 ug/paw) (FIG. 10).Thus, compounds that inhibit the synthesis or actions of linoleic acidmetabolites produce significant pain relieving effects in mammals.Moreover, at comparable dosages, these linoleic acid inhibitors producefar greater analgesia than morphine sulfate.

FIG. 11 depicts the effect of various oxidized linoleic acid metaboliteson the activity of TRPA1 or TRPM8 ion channels. The oxidized linoleicacid metabolites were found to be agonists for the TRPA1 ion channel.This ion channel is important for detection of pain and otherpathological states and therefore drugs that block the synthesis oractions of the oxidized linoleic acid metabolites may be useful fortreatment of pain, shock and inflammation via reduction of TRPA1activities. Whole cell patch clamp electrophysiological recordings ofCHO (Chinese Hamster Ovary) cells transfected with either TRPA1 or TRPM8show a striking response. The oxidized linoleic acid metabolites onlyactivated the TRPA1 channel. There was no effect on the TRPM8 channel.Several important conclusions can be made. First, the oxidized linoleicacid metabolites demonstrate selectivity, since some, but not all TRPchannels are activated. This is important in demonstrating a specificeffect and in establishing the non-obvious nature of the overallfindings. Second, the TRPA1 channel is strongly implicated in pain,whereas the TRPM8 channel is not. This indicates that drugs that blockoxidized linoleic acid metabolite synthesis or activity will have aselective effect on pain, shock or inflammation rather thannon-selectively altering all TRP channels.

FIG. 12 depicts the effect of lipids extracted from burned human skinsamples against cultured trigeminal sensory neurons from control (wildtype, “WT”) mice and from TRPV1 knock-out mice. Samples of burned humanskin and control (non-burned) human skin from patients undergoingtreatment in a hospital burn ward were obtained. The lipids from theskin samples were extracted by incubation with acetonitrile and thesamples dried under nitrogen gas. The samples were tested againstcultured trigeminal sensory neurons from control (wild type, “WT”) miceand from TRPV1 knock-out mice using real time imaging of calciumaccumulation (fura-2 method). Extracts from human control (non burned)skin had no effect on activating neurons from either control (WT) orTRPV1 knockout (TRPV1 KO) mice. However, the extracts from burned humanskin produced significant activation of neurons from WT mice. Thiseffect was reduced significantly reduced in the TRPV1 KO neurons,although about 50% of the activity was remaining. Thus, similar to theoxidized linoleic acid metabolites, the extracts from burned human skinactivate TRPV1 and some other channels.

FIG. 13 depicts the effect of lipids, extracted from burned human skinsamples, and then applied to CHO cells that expressed either TRPV1,TRPV2, TRPV3, TRPV4 or TRPA1, using real time calcium imaging. As shown,the extracts of the burned human skin activated TRPV1, TRPV3, TRPV4 andTRPA1, but not TRPV2. This shows that drugs that block the synthesis oractions of the oxidized linoleic acid metabolites would have strongeffects for treating pain, shock or inflammation in patients sufferingfrom pain conditions.

Collectively, these new data demonstrate the selectivity of the oxidizedlinoleic acid metabolites for activating TRP channels associated withpain, shock and inflammation, such as TRPV1, TRPV3, TRPV4 and TRPA1. Inaddition, the new data demonstrate that extracts from burned human skinin living patients activate sensory neurons via TRPV1 and activate CHOcells transfected with TRPV1, TRPV3, TRPV4 and TRPA1, suggesting thatoxidized linoleic acid metabolites active TRP channels associated withpain, shock and inflammation in humans. Thus, drugs that block thesynthesis or actions of these oxidized linoleic acid metabolites willhave utility for treatment of pain, shock and inflammation.

FIG. 14 depicts the effects of pretreatment with either cytochrome P450inhibitors (NDGA, DPI, CO or ketoconazole, ODYA), nitric oxide synthaseinhibitors (L-NAME, L-NNA), lipoxygenase inhibitors (BW-70C, PD, TEDC)or antioxidants (TROLOX, NAC, TEMPOL) versus negative controls (vehicle,argon) on the ability of linoleic acid to activate rat trigeminalsensory neurons as measured by real time levels of intracellular calcium(Fura-2, as indicated by the ratio of 340/380). The followingabbreviations are used:

-   -   1) NDGA: Nordihydroguaiaretic Acid    -   2) DPI: Diphenyliodonium    -   3) CO: Carbon Monoxide    -   4) 17-ODYA: 17-Octadecynoic acid    -   5) L-NAME: L-N^(G)-Nitroarginine methyl ester (hydrochloride)    -   6) L-NNA: N^(G)-nitro-L-Arginine; L-N^(G)-Nitroarginine    -   7) BW-70C:        N-[3-[3-(-Fluorophenoxylphenyl]-1-methyl-2-propenyl]-N-hydroxyurea    -   8) PD 146176: 6,11-Dihydro[1]benzothiopyrano[4,3-b]indole    -   9) 2-TEDC: 2-(1-Thienyl)ethyl        3,4-dihydroxybenzylidenecyanoacetate    -   10) TROLOX: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic        acid    -   11) NAC: N-Acetyl-L-cysteine    -   12) TEMPOL: 1-Oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine

The results indicate that the P450 inhibitors, including ketoconozole,and certain antioxidants, completely blocked the ability of linoleicacid to activate pain neurons. Although DPI and CO block all cytochromeP450s, the linoleic acid metabolism is a specific effect since moreselective P450 inhibitors (eg., ketoconazole vs ODYA) do not universallyinhibit the formation of these neuron-activating compounds. Thus,ketoconazole inhibits the formation of linoleic acid metabolites and isanalgesic, yet ODYA does not.

In this patent, certain U.S. patents, U.S. patent applications, andother materials (e.g., articles) may have been incorporated byreference. The text of such U.S. patents, U.S. patent applications, andother materials is, however, only incorporated by reference to theextent that no conflict exists between such text and the otherstatements and drawings set forth herein. In the event of such conflict,then any such conflicting text in such incorporated by reference U.S.patents, U.S. patent applications, and other materials is specificallynot incorporated by reference in this patent.

Further modifications and alternative embodiments of various aspects ofthe invention will be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as examples of embodiments. Elements and materials maybe substituted for those illustrated and described herein, parts andprocesses may be reversed, and certain features of the invention may beutilized independently, all as would be apparent to one skilled in theart after having the benefit of this description of the invention.Changes may be made in the elements described herein without departingfrom the spirit and scope of the invention as described in the followingclaims.

1. (canceled)
 2. A pharmaceutical composition for treating pain, shock,inflammatory conditions, or combinations thereof in a subject comprisingone or more compounds that inhibit and/or minimize the production ofoxidized linoleic acid metabolites and/or block the activity of oxidizedlinoleic acid metabolites and one or more pharmaceutically acceptablecarriers.
 3. The pharmaceutical composition of claim 2, wherein thepharmaceutical composition comprises one or more compounds that blockthe activity of oxidized linoleic acid metabolites, wherein at least oneof the compounds is an antibody that binds to at least one oxidizedlinoleic acid metabolite.
 4. The pharmaceutical composition of claim 3,wherein the antibody binds to a hydroxy linoleic acid metabolite.
 5. Thepharmaceutical composition of claim 3, wherein the antibody binds to anepoxy linoleic acid metabolite.
 6. The pharmaceutical composition ofclaim 3, wherein the antibody binds to an oxo linoleic acid metabolite.7. The pharmaceutical composition of claim 3, wherein the antibody bindsto one or more of: (10E,12Z)-9-oxooctadeca-10,12-dienoic acid;(9Z,11E)-13-oxooctadeca-9,11-dienoic acid; 9-hydroxyoctadecadienoicacid; 13-hydroxyoctadecadienoic acid; 9(10)-dihydroxy-octadec-12-enoicacid; 12,13-dihydroxy-9Z-octadecenoic acid; (12Z)-9,10-epoxyoctadecenoicacid; 12,13-epoxyoctadec-9Z-enoic acid.
 8. The pharmaceuticalcomposition of claim 3, wherein the antibody is a monoclonal antibody.9. The pharmaceutical composition of claim 3, wherein the antibody is apolyclonal antibody.
 10. The pharmaceutical composition of claim 1,wherein the pharmaceutical composition comprises one or more compoundsthat inhibit and/or minimize the production of oxidized linoleic acidmetabolites.
 11. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition comprises one or more compounds that inhibitand/or minimize the production of oxidized linoleic acid metabolites,wherein at least one of the compounds is a cytochrome P-450 enzymeinhibitor.
 12. The pharmaceutical composition of claim 9, wherein thecytochrome P-450 enzyme inhibitor is ketoconazole.
 13. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition comprises one or more compounds that inhibit and/or minimizethe production of oxidized linoleic acid metabolites, wherein at leastone of the compounds is an antioxidant sufficient to substantiallyinhibit and/or reduce the catalytic effect of relevant metabolic enzymesin the Linoleate pathway. 12-24. (canceled)
 25. The pharmaceuticalcomposition of claim 1, wherein the composition comprises ketoconazoleand nordihydroguaiaretic acid.
 26. pharmaceutical composition of claim1, wherein the pharmaceutical composition comprises up to about 10% byweight of ketoconazole.